The death receptor-induced apoptosis of tumor cells by TRAIL or agonistic antibodies is thought to be an important emerging strategy for cancer therapy. We have developed an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our pre-clinical studies have demonstrated its strong anti-tumor efficacy and safety in animal models, and Phase I clinical trials are planned. However, both pre-existing and induced resistance of tumor cells to DR5-mediated apoptosis is a concern. We have identified a RNA helicase of the DEAD box protein family, DDX3, which serves as a critical adaptor protein in regulation of DR5 signaling transduction, and plays a causative role in induction of DR5 apoptosis resistance. The overall goal of this proposal is to examine the role of DDX3 in the development of resistance to DR5-mediated apoptosis. The central hypothesis is that DDX3, functioning as an adaptor protein, is constitutively associated with DR5 via a specific binding motif in each molecule. Near its N-terminus, DDX3 recruits clAP1 via a CARD/CARD interaction between the two molecules. Thus, a default function of the DR5/DDX3/clAP1 is to negatively regulate DR5-mediated apoptosis. In DR5 apoptosis sensitive cells, activation of the initiator caspase 8 leads to cleavage of DDX3 at aa135, which releases the N-terminal CARD of DDX3 and clAP1 from DR5/DDX3/clAP complex, thereby enabling a positive feedback loop to amplify apoptosis signal. In contrast, in DR5 apoptosis resistant cells, increased recruitment of clAP1 leads to inhibition of caspase 8 activity and failure of DDX3 cleavage, thereby forming a negative feedback loop to prevent amplification of the initial apoptosis signal. The Aims to test four hypotheses are: 1) that the association of DDX3 with DR5 is essential;2) that the clAP1 recruited by a CARD of DDX3 is a key inhibitory molecule in the initiation of DR5-mediated apoptosis;3) that the caspase-mediated cleavage of DDX3 releases the N-terminal CARD from DR5 thereby reversing the resistance;and 4) that the interruption of the DR5/DDX3/clAP1 complex may improve the therapeutic efficacy of TRA-8 and other DR5-directed agents. The proposed studies will provide novel insights into the role of DDX3 in DR-5 mediated apoptosis, and also will have implications for the further development of interventions to enhance the therapeutic efficacy of TRA-8 and other agonistic DR5 antibodies and TRAIL.